滲透壓和Aurintricarboxylic Acid對中國倉鼠卵巢細胞生理之影響

Abstract

葡萄糖是哺乳動物細胞重要的碳源，提供細胞生長、合成、進行生化反應能量，為了解葡萄糖造成之滲透壓對於Chinese hamster ovary (CHO) K1 cell生長影響，本論文於MEM培養基分別添加9 g/L葡萄糖或9 g/L 山梨醇濃度提高培養基之滲透壓來培養細胞，結果發現滲透壓會影響細胞生長速率，山梨醇造成細胞生長速率延緩具有顯著差異( P < 0.05 )，顯示高滲透壓會延緩細胞生長。西方墨點實驗結果顯示高滲透壓在第1小時可引起CHO K1 cell p38 MAPK磷酸化，及在第1、15及30小時，引起p42 MAPK活化，並推測高滲透壓造成細胞週期延緩可能與p38 MAPK活化有關。

在探討Aurintricarboxylic acid (ATA)濃度對CHO β-gal serum free (SF) cell造成細胞貼附(adhesion)與細胞間交互作用(aggregation)改變方面，發現低濃度(30 ppm) ATA可促使部分CHO β-gal SF cell由懸浮貼附至培養皿成梭狀，高濃度(100 ppm) ATA可影響CHO β-gal SF cell由懸浮聚集變成不聚集狀態。以免疫螢光染色法分別偵測細胞cadherin與integrin β1表現情形，實驗結果發現ATA濃度不會影響cadherin表現，但CHO β-gal SF cell integrin β1表現會隨著ATA濃度增加而增加且具有顯著差異( P < 0.05 )，不同濃度ATA引起細胞貼附至培養皿或變成不聚集狀態，其機制可能是經由integrin表現而調控造成。

Glucose is an essential carbon source for cell growth and biochemical synthesis in mammalian cells. To study the effects of glucose on Chinese hamster ovary (CHO) K1 cell, extra 9g/L glucose or 9 g/L sorbitol were added to MEM medium to increase osmotic stress. The results showed cell growth rate decreased with hyperosmotic stress, while sorbitol had a strong effect on reducing CHO K1 cell number (P < 0.05). Hyperosmotic stress induced p38 MAPK phosphorylation at the first hour, and also activated p42 MAPK at first, fifteenth, thirtieth hour. It suggested that the cell growth rate decreased might be caused by p38 MAPK activation.

Aurintricarboxylic acid (ATA) effects on cell morphology and cell-cell interaction were examined. We found that 30 ppm ATA caused CHO β-gal serum free (SF) cell to adhere, but 100 ppm ATA affected CHO β-gal SF cell aggregation and resulted in non-aggregation. Cadherin and integrin β1 expression of CHO β–gal SF cell were detected by immuno-fluorescence. The results showed cadherin expression was unaffected by ATA concentration, whereas low ATA concentration (30 ppm) stimulated integrin expression (P < 0.05) and high ATA concentration (100 ppm) induced integrin overexpression (P < 0.05). The stimulation of adhesion and inhibition of aggregation may be regulated by integrin mediated signal pathways.